Aspirating and mixing of liquids within a probe tip

ABSTRACT

Apparatus and a method for mixing a liquid within a disposable aspirating probe tip so that most of the liquid is forced to move past a transition zone between two different inside diameters to cause rotational mixing. The apparatus and method can be used to provide agglutination of blood, which in turn can be used for blood typing. The probe tip can comprise a single integral piece, or two separate portions.  
     The transition zone can comprise a sharp demarcation between inside diameters, or a smooth one.

FIELD OF THE INVENTION

[0001] The invention relates to apparatus and a method for mixing twoliquids within a tip on an aspirating probe, to ensure a reactionbetween the liquids.

BACKGROUND OF THE INVENTION

[0002] It is known from U.S. Pat. Nos. 5,773,305 and 5,114,162 to mix afluid sample such as blood and a diluent, inside a probe tip by firstaspirating both liquids into the tip, and then drawing said liquidsfurther up into the tip into a mixing chamber having an enlarged insidediameter compared to the rest of the tip. The mixing can be achieved,for example, by reciprocating the mass of liquids up and down numeroustimes.

[0003] In the examples shown in U.S. Pat. No. 5,773,305, the liquids areretained in the enlarged chamber and simply sloshed back and forth inthat chamber to achieve mixing. FIG. 3 thereof makes it clear thatsimply aspirating the liquids into the enlarged chamber past a stepdiscontinuity created by the enlarged inside diameter, is ineffective increating a mixture. That is, a single movement past the stepdiscontinuity is shown as not mixing the fluids homogeneously. An airbubble can also be included between the liquids when first aspirated.Cross-over contamination between bodies of liquid being aspirated ispreferably prevented by ejecting an inert oil shield around the outsideof the tip, FIGS. 7 through 11 thereof.

[0004] Such a construction is generally equivalent to transferring twoliquids from a pipette into a larger diameter container (the mixingchamber) and attempting mixing by sloshing the liquids vertically withinthe container. Although mixing can occur in such a fashion forrelatively large volumes, it is not as effective for small volumes,e.g., volumes that total 100 to 600 microliters. That is, in a constantdiameter channel, inertial mixing is reduced if the volumes are small ashere. It is this phenomenon that requires the movement of the liquidsback and forth in the mixing chamber, as much as 20 times, to achievehomogeneous mixing. Such reiterations of the mix step aretime-consuming, and beg for an improvement.

[0005] Furthermore, it is not the case that cross-contamination ispreventable only by using such an oil shield. That is, in some cases,the first-aspirated liquid can be removed from the tip simply berepeated washing with a diluent, or by wiping. In any event, shouldwashing prove to be unsatisfactory, there has been a need for a morereliable method of preventing contamination than by using the oilshield. (The oil shield is not guaranteed to form completely around thetip just because a plural of dispensing nozzles are disposed about thecircumference of the exterior of the tip.) Furthermore, some proteinscan destroy the shield effect of the oil.

[0006] In the examples of U.S. Pat. No. 5,174,162, all the liquids to bemixed are moved completely into the enlarged mixing chamber, completelyout of the chamber, then back into it, and so forth. The sharptransition at surface 15 causes turbulent mixing, 16, FIG. 2 thereof.This is a more efficient mixing method than that of the '305 patent.Nevertheless, there are improvements that are needed in such a mixingsystem as described in the '162 patent. For example, no option isdescribed for the geometry of FIG. 2. Nothing is described regarding anyuse of air bubbles to separate the liquids as they are aspirated. Asnoted however in the '305 patent, such an air bubble provides aneffective prevention against cross-contamination. Yet, any air bubblemust be rapidly eliminated during mixing.

[0007] Furthermore, the '162 patent is notably deficient in any teachingto prevent cross-contamination when aspirating liquid 6 immediatelyafter liquid 4, between the two liquids within the bulk container ofliquid 6. Although the oil shield of the '305 patent might seem to beapplicable to the probe of the '162 patent as well such a shield hasdisadvantages as noted above. Alternative protection methods againstcross-contamination, besides the oil-shield method, are thus desirable.

[0008] Yet another disadvantage of the teachings of the '162 patent isthat when the two disparate liquids are moved back and forth across theboundary 15, unmixed “tails” of one or both liquids can be left behindas coatings on either the enlarged chamber or the narrower intakeportion. Such residual tails do not get mixed when the main body ofliquids is moved across boundary 15, so that the tails are undesirable.

[0009] Thus, although substantial development has already occurred inprobes designed to mix two liquids entirely with the probe, thereremains the need for improvements.

SUMMARY OF THE INVENTION

[0010] We have devised a mixing method and a probe tip for doing themixing therein, that provide the above-noted needed improvements.

[0011] More specifically, in accord with one aspect of the invention,there is provided a method of mixing a plurality of liquids, comprisingthe steps of:

[0012] a) providing a probe tip with an internal cavity having aplurality of different inside diameters;

[0013] b) providing by aspiration a plurality of liquids inside aportion of the probe tip;

[0014] c) moving at least most of said liquids back and forth at leastseveral times between a part of said cavity with a smaller insidediameter and a part with a larger inside diameter, said larger andsmaller diameters being sufficient to provide a sufficient rotation ofliquid as it moves between diameters to cause mixing of said liquids;

[0015] the improvement wherein the capillary number resulting from themixing in step c) does not exceed about 0.01, the capillary number beingdefined as the ratio of liquid velocity times viscosity and surfacetension, so that any tails formed during the mixing step c) areminimized.

[0016] In accord with another aspect of the invention, there is provideda method of mixing a plurality of liquids comprising the steps of a)through c) listed above, wherein the improvement comprises that thecavity parts comprise two separate but matable tip portions, and themethod further includes the step of mounting a tip portion of one of theinside diameters onto the tip portion of the other inside diameterin-between aspiration of liquids, such that carry-over contaminationbetween liquids is prevented.

[0017] In accord with still another aspect of the invention, there isprovided a method of mixing a plurality of liquids comprising the stepsof a) through c) listed above, wherein the improvement comprises theinside diameters each provide a cross-sectional flow-through area of thecavity part, and the cross-sectional flow-through area of the largerinside diameter is at least three times the cross-sectional flow througharea of the smaller inside diameter, for maximum mixing efficiency.

[0018] In accord with yet another aspect of the invention, there isprovided a method of mixing a plural of liquids comprising the steps ofa) through c) listed above, wherein the improvement comprises the largerinside diameter being obtained by i) selecting as a first tip portion atapered tip at least a portion of which has an inside diameter that ismuch larger than the smaller inside diameter of the probe tip, and ii)joining the tapered tip to the probe tip having the smaller insidediameter.

[0019] In accord with yet another aspect of the invention, there isprovided a method of mixing a plurality of liquids comprising the stepsof a) through c) listed above, wherein the improvement comprisesproviding a total amount of liquid in step b) such that if all liquid ismoved into the part with the larger inside diameter, the larger insidediameter is greater than the height of the total liquid, but less thantwice the height of the total liquid, so that mixing as per step c) ismaximized.

[0020] In accord with yet another aspect of the invention, there isprovided a method of mixing a plurality of liquids comprising the stepsof a) through c) listed above, wherein the improvement comprises movingin the step c) at least most of the liquids back and forth at leastbetween the cavity part with the smaller inside diameter and a part ofthe cavity of a larger inside diameter located at opposite ends of thecavity part of the smaller inside diameter, so that mixing efficiency isenhanced by rotation of the liquid as it moves past the opposite ends,rather than a single end of the smaller inside diameter cavity part.

[0021] In accord with yet another aspect of the invention, there isprovided a method of mixing a plurality of liquids comprising the stepsof a) through c) listed above, wherein the improvement comprises movingin the step c) at least most of the liquids back and forth at leastbetween the cavity part with the smaller inside diameter and a part ofthe cavity of a larger inside diameter located at opposite ends of thecavity part of the smaller inside diameter, so that mixing efficiency isenhanced by rotation of the liquid as it moves past the opposite ends,rather than a single end of the smaller inside diameter cavity part.

[0022] In accord with yet another aspect of the invention, there isprovided a probe tip for liquids within the tip after aspiration of theliquids therein to, the tip comprising

[0023] a wall defining 3 connected cavities of unequal inside diametersone of the compartments being sandwiched as a middle compartment betweenthe other two which form end compartments, each two adjacent cavitiesbeing connected by a transition zone wall and the inside diameters beingsufficiently unequal in the adjacent 2 cavities as to cause rotationalmixing of liquids as they move past the transition zone wall,

[0024] wherein the transition zone of the one cavity is formed by avariance of the inside diameter that increases in value as themiddlemost cavity is transited outward into either of the other two endcavities.

[0025] In accordance with yet another aspect of the invention, there isprovided a method of determining the strength of an agglutinationreaction within a hollow container comprising walls capable oftransmitting light at certain predetermined wavelengths, comprising thesteps of:

[0026] a) providing a mixture of a sample and an agglutinating reagentwithin a first cavity of the container, the cavity having a first insidediameter,

[0027] b) transferring the mixture to a second cavity having a secondinside diameter substantially smaller than the first inside diameter,

[0028] c) scanning the liquid within the second cavity during the stepb) with a beam of light at the predetermined wavelengths, the 10%portion being that portion closest to the first cavity;

[0029] d) after the scanning step c), detecting the amount of lightabsorbed within or scattered by the 10% portion by the beam,

[0030] e) transferring the mixture back into the first cavity,

[0031] f) repeating steps b)-d) at least once until some agglutinatedmaterial has separated from non-agglutinated material, and

[0032] g) calculating the amount of agglutination from the absorbance orscattering detected in step d).

[0033] In accordance with yet another aspect of the invention, there isprovided a method of agglutinating blood cells in whole blood,comprising the steps of

[0034] a) aspirating whole blood into a disposable tip mounted on aprobe, said tip having at least two portions with significantlydifferent inside diadems, connected to each other by a transition zone,

[0035] b) aspirating into the same tip thereafter, an agglutinatingreagent, and

[0036] c) moving said blood and reagent back and forth as a totalliquid, first entirely into one of said portions and then entirely intothe other of said portions, a sufficient number of times so as to causecoagulation of the cells of the whole blood, and then subsequentseparation of plasma from the coagulated cells.

[0037] As used herein, “probe tip” or “probe tip portion” means anyvessel, disposable or not, into which liquid can be aspirated, mountableon an aspirating probe, that comprises the features noted, namely anorifice, an interior chamber spaced from the orifice, and a passagewayconnecting the orifice and the chamber. Thus, the tip or tip portion canbe a conventional disposable tip such as is shown in U.S. Pat. No.4,347,875 by Columbus, or even a cup or well with an orifice in thebottom, such as the cup shown in U.S. Pat. No. 5,441,895 but with anorifice in the bottom. The tip can be one integral piece or provided inseveral portions.

[0038] Accordingly, it is an advantageous feature of the invention thatmore rapid mixing of two liquids aspirated into the tip, takes placewithin the tip than occurs with conventional devices.

[0039] It is a related advantageous feature of the invention that noadditional device is needed beyond the tip that is used anyway foraspiration, to provide mixing.

[0040] It is another advantageous feature of the invention that, in someembodiments, carryover contamination between liquids aspirated ispreventable by an inexpensive mechanical device that is less timeconsuming than repeated washing.

[0041] A related advantage of the aforesaid mechanical device forpreventing carry-over contamination, is that it renders the tip of theinvention more manufacturable.

[0042] Other advantageous features will become apparent upon referenceto the Detailed Description of the Embodiments, when read in light ofthe attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 is a fragmentary elevational view in section of a probe tipconstructed in accordance with the prior art;

[0044] FIGS. 2A-2C are fragmentary elevational views in section, similarto that of FIG. 1, but illustrating a method of the invention;

[0045] FIGS. 3-5 are fragmentary elevational views similar to that ofFIG. 2, but illustrating certain preferred embodiments;

[0046]FIG. 6A is a fragmentary elevational view similar to FIGS. 2-5,except it illustrates an alternative embodiment wherein the second tipportion that is added between aspirations, has a narrower insidediameter than the first tip portion;

[0047]FIG. 6B is a view similar to that of FIG. 6A, showing thesubsequent steps of mixing,

[0048]FIG. 6C is an elevational view similar to that of FIG. 6A, but ofan alternate embodiment;

[0049] FIGS. 7A-7H are elevational views in section similar to FIGS.6A-6C, except showing a further additional embodiment wherein liquidflowing from the second tip portion to the fast tip portion isconstrained to move into a narrower, rather than wider, diameter formixing;

[0050]FIGS. 8 and 9 are elevational views in section similar to that ofFIG. 4, but showing still further embodiments of the invention; and

[0051]FIGS. 10 and 11 are plots of absorbance versus amount of liquidscanned by a light beam scanning through the tip, to illustrate a methodof detecting the strength of an agglutinating reaction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The invention is hereinafter described in connection with certainpreferred embodiments, wherein mixing of one or two liquids, one ofwhich is body liquid, is achieved using a disposable tip with one or twoportions of preferred shapes, the second being preferably separate fromand added to the first to prevent carry-over contamination of a thesecond liquid after the first liquid is aspirated, wherein the firstliquid is preferably blood and the second is an agglutinating solution,and mixing is accomplished at preferred flow and shear rates, preferablyto allow blood typing to occur. In addition, the invention is usefulregardless of how many and what shape portions the tip is divided into,whether a second portion is separately added or already present, or isused to prevent contamination or not, what the liquid compositions are,what order they are added, what the flow and shear rates within the tipare, and what the end result of the mixing is; provided that the tip apeinduces mixing by forcing the mixing liquids to move between cavityparts with differing diameters sufficient to cause rotational mixing ofthe liquids as they flow between the cavity parts. That is, it isrepeated movement between the transition in diameters that causes rapidmixing, rather than sloshing the liquids within a constant insidediameter. Thus, the reagent interacting with the body liquid can bereagents for an immunoassay, for example.

[0053] The less successful approach is that of the prior art, shown hereas FIG. 1. This is substantially the teaching of the aforesaid U.S. Pat.No. 5,773,305. In such an arrangement, an aspiration probe 12 comprisesa narrow cavity or passageway 14 that leads from an aperture 34 at end36, to a mixing cavity 18 having a significantly wider inside diameterthan that of cavity 14. A transition region 28 with relatively sharpdemarcations is provided between the two diameters. A partial vacuum isapplied at passageway 40 to aspirate first a liquid 44, and then asecond liquid 54, into cavity 18, with or without a bubble (not shown)between them. As shown in the original '305 patent, the first time bothliquids are moved from passageway 14 into cavity 18 fails to producecomplete mixing, since the two liquids still remain separated The actualmixing is achieved by oscillating the two liquids, arrows 30, withincavity 18, from an end at transition zone 28, to the opposite end 32 ofcavity 18. Additionally, an oil shield is taught as useful on theexterior surface 36 of the probe, to prevent liquid 44 from coatingsurface 36 and contaminating bulk liquid 54 when the latter isaspirated. As noted above, such a techniques requires as much as 20oscillations, arrows 30, to achieve mixing.

[0054] In both the '305 patent and the instant invention, movement ofliquids within the tip is achieved while the tip is on a pipette, byactuation of a piston within a piston cylinder, not shown, to create apartial pressure or partial vacuum. For example, the piston can beoperated manually.

[0055] In accordance with the invention, FIGS. 2A-2C, the number ofoscillations can be reduced to as few as three, by simply forcing mostof the liquid to flow past the transition zone 128 each time. This, inturn, is ensured by forcing most of both liquids to flow from one cavityadjacent the transition zone, into the other cavity so adjacent, andthen back As used herein, “most of the liquids” being moved means, atleast 90% of the liquids.

[0056] More specifically, a probe preferably in the form of a disposabletip 112, is constructed substantially the same as that of the prior art,with a narrower cavity 114 leading to a wider cavity 118 connected tothe narrower one by a transition zone 128. A common axis of symmetry 100preferably extends through both cavities.

[0057] In this example, the transition zone is defined by relativelysharp edges 134 and 136 at the junction with the respective cavities.“Relatively sharp” means, having a radius of curvature at the junctionthat is less than 25 microns. Any radii greater than that tend toproduce a smooth transition between zone 128 and the respectivecavities. In fact, a smooth transition through the use of such greaterradii of curvature is preferred, but not essential, as such a smoothtransition gives better results when blood agglutination for bloodtyping is the goal of the mixing. That is, the smooth transition usinggreater radii of curvature is less likely to cause the agglutinates tobe broken up, all other things such as bulk flow rates, being equal. Anexample of a smooth transition using such greater radii of curvature isshown in FIG. 4. For example, R₁, and R₂ for FIG. 4 can be,respectively, 1.2 mm each.

[0058] Since the structure is generally the same as for FIG. 1 of theprior art, the main distinction, at least with respect to FIGS. 2A-2C,is in the use of probe 112. That is, a first liquid 144 is aspiratedinto cavity 114, followed by an air bubble 160. Thereafter, secondliquid 154 is aspirated in so that both are still in cavity 114, FIG.2A.

[0059] Next, most and preferably all of both liquids are aspirated pastzone 128 and into cavity 118, FIG. 2B. Transition zone 128 producessufficient rotation, arrows 170, of the liquids as to start them to mix.As shown in FIG. 1, however, just this step is not enough. Next, mostand preferably all of the liquids are ejected from cavity 118 pasttransition zone 128 and into cavity 114, arrows 172, FIG. 2C. Stillfurther, the process is repeated, phantom arrows 174, until completemixing has occurred. Depending on the liquids involved, only threepassages from cavity 114 into cavity 118 may be necessary for completemixing, although more can be used.

[0060]FIG. 3 illustrates certain preferred parameters for optimal mixingin general. Probe 112 has an aperture 134 and an exterior surface 136adjacent to that aperture, similar to that of the prior art. However,the cross-sectional flow-through area A₂ of cavity 118, provided byinside diameter D₂, is preferably no smaller than nine times that of thecross-sectional flow-through area A₁ provided by inside diameter D₁, ofcavity 114. Furthermore, the diameters D₁ and D₂ are generally constantso that their respective cavities are cylindrical Thus, D₂ is preferablyat least equal to three times D₁.

[0061] Useful examples of D₁ and D₂ include, e.g., 0.8 mm and 3.2 mm,respectively, for use with a total height H₂, FIG. 5, of about 3 mm.

[0062] Still further, to aid in the dispersal of air bubble 160, FIG.2A, during mixing, the wall surface of at least cavity 118, andoptionally also cavity 114, is selected from materials that are easilywetted by the liquids in question, that is, produce a low contact angleat the meniscus. Thus, the materials used for the surfaces are afunction of the liquids to be mixed, as is well-known. Most preferably,for maximum dispersal of the air bubble (present to aid in preventingcross-contamination between liquids during the second aspiration), thecapillary number for the system does not exceed 0.001, where capillarymember, as is conventional, equals liquid velocity of movement, arrow170, divided by surface tension of the liquid mixture.

[0063] However, it is not essential that an air bubble be present toavoid contamination. An oil shield can be used as in the '305 patent, oralternatively, probe 112 can be wiped off before aspirating the secondliquid. In that case, the capillary number can be larger, but preferablynot exceeding 0.01, since above that, the movement of the liquidsbetween cavities can product “tails” of liquid remaining in the exitcavity that delay or even ruin the mixing process.

[0064] If an air bubble is used, a further consideration is that thesize or volume of the bubble must be less than that which will preventmixing of the liquids as they flow past the transition zone. Thus, theair bubble must not be so large that, after aspiration of the probecontents into cavity 118, FIG. 3, the bubble (not shown in FIG. 3)continues to totally separate the two liquids—that is, has a diameterequal to the inside diameter of cavity 118. Thus, for FIG. 3, the bubblevolume must be less than π(D₂)³/6.

[0065] In the event the mixing is being done for blood typing, a furtherfactor is important in addition to those noted above. That is, toprevent the rotational action, arrows 170, FIG. 3, from significantlybreaking apart the desired blood cell agglutination, the flow velocityin either direction past the transition zone 128 is preferably thatwhich provides a shear rate along the wall which does not exceed about20 sec⁻¹. This, of course, is also a function of the viscosity of theliquids, of the diameters D₁ , D₂, and of angle alpha.

[0066] Regardless of the end use of mixing, the embodiment of FIG. 3 canalso be used by coating either cavity 114 or 118 in dry form, with thereagent that is to react with the body liquid, so that only one liquidnamely the body liquid, need be aspirated in at aperture 134. Thus, theagglutinating reagent solution can be provided during manufacturing bycoating either or both cavities 114 or 118. This coating is thenredissolved when the whole blood is aspirated into the appropriatecavity.

[0067] For other uses, other reagents, such as an antibody for animmuno-assay, may be permanently attached to the cavity walls.

[0068] It is not essential that the probe be al in one piece, or thatcontamination be prevented by only an oil shield or by wiping. Instead,FIG. 4, it is useful to have the probe comprise two portions, 112A and112B one of which (112B) has an inside diameter that is different from,e.g., smaller than, at least part of the inside diameter of the otherportion (112A), and which fits over the other portion adjacent aperture134. The purpose is to allow the portion that over-fits the fastportion, to cover up the exterior surface 136 adjacent to aperture 134where residual first liquid 44 might remain. As shown, inside diameterD₃ of cavity 165 of portion 112B is substantial identical to diameterD₁, but less than diameter D₂, of portion 112A. In use, liquid 44 isaspirated into portion 112A with portion 112B absent. Portion 112B isthen mounted onto portion 112A with a sliding fiction fit. At thispoint, liquid 44 is moved down, arrow 180, into portion 112B to thephantom position 182, leaving an amount of air at 160 to form an airbubble in the next step. That step is to move the probe of combined tipportions so as to insert only portion 112B into a bulk quantity ofliquid 54 (not shown). Aspiration then causes liquid 44 at position 182,bubble 160, and an amount of the second liquid to be aspirated in theprobe. When the desired amount of the second liquid is present, theprobe is removed from the bulk liquid 54, and mixing proceeds asdescribed above using repeated movement of most of the liquids pasttransition zone 128.

[0069] This construction ensures both that residual first liquid amountson portion 112A are prevented from contacting said bulk liquid 54, andthat the results length of portions 112A and 112B are easily moldable.

[0070] In this embodiment, and any embodiment using a smooth transitionbetween zone 128 and the two cavities provided by the radii of curvatureR₁ and R₂, angle alpha described above is measured against the tangentline A-A drawn to a point on the wail of zone 128 that is between thedefinition of the wall provided by the two radii.

[0071] In FIG. 5, another preferred aspect of the probe 112 isillustrated. That is, cavity 118 of portion 112 or 112A has a diameterD₂ that is selected to be larger in value than the height H₂ of thetotal liquids aspirated thereinto, one those liquids have been movedinto cavity 118. The advantage of this relationship is that it has beenfound to enhance the mixing efficiency. At the same time, however, D₂should be less than twice H₂, as otherwise the volume in cavity 118becomes so thin that it is in danger of bursting at the middle whenpressure is applied to push the liquid, arrow 200, into cavity 114. Suchbursting will of course prevent transfer of the liquid across the mixingtransition zone.

[0072] Thus, if all of the preferred features are utilized as describedabove, it has been found that a whole blood sample and an agglutinatingsolution can be thoroughly admixed after only three cycles of drawingmost of the liquids into cavity 118 and returning most of the liquids tocavity 114.

[0073] As noted above, when the probe comprises two portions, it is notessential that the inside diameter of the added-on portion equal theinside diameter of the probe portion that is covered The remainingembodiments illustrate wherein, in fact, this is not the case. Partssimilar to those previously described bear the same reference numeral towhich the distinguishing mark ‘ or ” is appended.

[0074] Thus, in the embodiment of FIG. 6A, the second portion 112B′ hasan inside diameter D₃ for cavity 165′ that is considerably smaller thaninside diameter D₂ of cavity 118′. In effect, probe 112′ is now dividedinto two separable portions 112A′ and 112B′ having cavities 118′ and165′ which between them provide the transition zone 128′ that causesmixing. That is, zone 128′ is formed by an external angle alpha (shownin FIG. 5) which is 270 °.

[0075] In use, liquid 44′ is aspirated into portion 112A′ by itself.Portion 112B′ is then affixed to portion 112A′ as shown, FIG. 6A, andliquid 44′ is pushed down into portion 112B′, arrow 200. The probe isthen moved so that portion 112B′ is inserted into a bulk quantity ofliquid 54′, preferably with an air bubble 160′ at aperture 134′, FIG.6B. Aspiration, arrow 202, causes all of liquid 44′, bubble 160′, andliquid 54′ to move through cavity 165′, past transition zone 128′, andinto cavity 118′, thus starting mixing by rotation, arrows 170′. Theoscillating movement of all the liquid via arrows 200 and 202 is thenrepeated as many times as is needed to complete the mixing.

[0076] Alternatively, FIG. 6C, the transition zone provided by theadd-on portion 112B′, when placed around exterior surface 136A′ adjacentaperture 134A′, can be a smooth transition zone 128′ in the manner ofthe embodiment of FIG. 4. In such a case, care needs to be taken toensure that a proper match of the inside diameters of portions 112A′ and112B′ occurs at aperture 134A′, so that indeed the transition in insidediameters is a smooth one. At the same time, however, the smaller insidediameter remains with probe portion 112B′, rather than portion 112A′,except where they match substantially exactly at aperture 134A′.

[0077] The opposite of FIGS. 6A and 6B is illustrated in FIGS. 7A-7H.That is, the inside diameter of the added-on, second tip portion issubstantially larger, at the transition zone, than the inside diameterof the first tip portion already used to aspirate liquid. Additionally,this embodiment illustrates that the two cavities adjacent thetransition zone need not be cylindrical, but can be tapered insteadalong their axis of symmetry 100, FIG. 7B.

[0078] Thus, FIG. 7A, probe portion 112A″ comprises a conical cavity118″ extending from an aperture 134A″, to an upper portion 132A″ thatconnects to a pump, not shown, the inside diameter of cavity 118″increasing with increasing distance from the aperture. To allow the twoportions 112A″ and 112B″ to join together, the exterior surface 136A″adjacent to aperture 134A″ is enlarged, also with a tapered shape, suchas by securing a cork collar to the rest of the portion 112A″. Theinside diameter at aperture 134A″ is relatively small, e.g., about 1 mm.

[0079] The second probe portion 112B″, FIG. 7B, has an upper portion132B″ shaped to frictionally mate with surface 136A″, that is, with anenlarged inside diameter. Portion 112B″ tapers down to a lower portionat aperture 134B″ producing a cavity 165″ having an inside diameter thatis greatly reduced from said enlarged inside diameter, and in fact,preferably is about the same as that of aperture 134A″.

[0080] The use of this embodiment is similar to that described for FIGS.6A-6B. Thus, portion 112A″ by itself is inserted into a bulk quantity ofliquid 44″ and an aliquot is aspirated, FIG. 7A. Next, probe portion112B″ is fitted over the surface 136A″ of the collar, FIG. 7B. Afterthat, liquid 44″ is pushed or ejected from portion 112A″ into the cavityof portion 112B″, FIG. 7C.

[0081] Next, FIG. 7D, the combined probe has aperture 134B″ of portion112B″ inserted into a bulk quantity of liquid 54″, and that and an airbubble 160″, FIG. 7E, is aspirated into cavity 165″.

[0082] The stage is now set for the actual mixing steps. That is, FIGS.7F-7H, all of the liquid is aspirated and ejected back and forth pastthe transition zone created by the narrower inside diameter at aperture134A″. FIG. 7F, it is first drawn into cavity 118″, arrow 202″, toproduce the condition shown in FIG. 7G. It is then ejected back intocavity 165″, FIG. 7H, arrow 200″, so that rotational mixing occurs. Thisprocess is repeated as necessary, until the two liquids becomehomogeneous, or as homogenous as is possible, given the nature of theliquids.

[0083] In all of the embodiments above wherein a second probe portion112B is fitted onto the first portion 112A prior to aspirating a secondliquid, another alternative, following such second aspiration andaspiration of all liquids into the first portion, is to remove thesecond portion and to fit onto the first portion in the place of thesecond, a clean third portion of equal, smaller, or larger insidediameter, for the purpose of aspirating into the probe yet another,third liquid in a manner similar to the aspiration of the second liquid.

[0084] Additional mixing transitional zones between unequal insidediameters can be provided—that is, it is not essential that there beonly two adjacent compartments of varying inside diameters. Indeed, aprobe tip that comprises three such compartments serially connected,FIGS. 8-9, has proven to be most efficient in mixing, of all theembodiments described herein. Most preferably, in such an arrangementthe middlemost compartment has the smallest inside diameter at thetransition zone. Parts similar to those previously describe bear thesame reference numeral, to which the distinguishing superscript suffix′″has been appended.

[0085] Thus, FIG. 8, like the design of FIG. 4, probe 112′″ comprises anupper portion or cavity 118′″ that is mounted onto the permanent probe(FIG. 4), and a lower cavity 114′″ integrally connected to cavity 118′″by a transition zone wall 128 , the inside diameter D₂ of cavity 118being larger than D₁, and preferably at least equal to three times D₁.An additional cavity 165 is provided at exterior portion 136′″ of cavity114, with aspiration occurring at arrow 210, also as described for FIG.4. However, cavity 165′″ is integrally connected to cavity 114′″ in thatall 3 cavities are formed from a common wall, preferably one that ismolded. Further, inside diameter D₃ of cavity 165′″ is significantlylarger than inside diameter D₁, creating a transition zone 220 notpresent in the embodiment of FIG. 4. The value of D₃, like that of D₂,is selected to cause rotational mixing when most, and preferably all, ofthe liquids aspirated into tip 112′″, is moved from cavity 114′″ intocavity 165′″ past transition zone 220. Hence, like D₂, D₃′ is mostpreferably at least equal to three times D₁. D₃ can be the same as ordifferent from D₁.

[0086] Although it is not essential, the inside diameter of cavity 165′″can be narrowed to D₃ at the end into which liquid is first aspirated,arrow 210.

[0087] Additionally, as shown in FIG. 9, cavity 165′″ of tip 112′″ canbe formed by the wall of tip portion 112B′″, removable as in theembodiment of FIG. 4, so that portion 112B′″ can be added after thefirst liquid is aspirated, and portion 112B′″ covers any first liquidremaining on exterior surface 136′″. Aspiration of a second liquid thenoccurs as shown by arrow 210′″, FIG. 9. However, unlike the FIG. 4embodiment, inside diameter D₃′ at the junction of the cavities 114′″and 165′″, is greater than the inside diameter D₁, rather than equalthereto as in FIG. 4, creating a transition zone 220′″ similar to zone220, FIG. 8, effective to cause liquids to rotationally mix as they movefrom cavity 114′″ into cavity 165′″. In this example, D₃′ at thetransition zone equals D₁+twice the value of T, where “T” is thethickness of the wall providing exterior surface 136′″. In such anexample, D₃′ may or may not be at least equal to three times D₁,depending on the value of T.

[0088] As in the case of the embodiment of FIG. 8, the inside diameterof cavity 165′″ can be narrowed to D₃″ a the end into which liquid isfirst aspirated.

[0089] It is the embodiments of FIGS. 8 and 9 that have proven to bemost efficient in mixing, that is, in producing complete mixing in thefewest cycles of repeated back and forth movement past the transitionzones. For example, the embodiment of FIG. 9 produced complete mixing oftwo liquids totaling 20 microliters in only 7.5 cycles of such back andforth movement, at a flow rate of 50 microliters per sec., in about 10sec.

Agglutination Reactions

[0090] As noted above, a preferred use of this mixing action is toproduce sufficient blood cell agglutination as to allow blood typing. Tothat end, one of the liquids is, of course, whole blood and the other isa solution of agglutinating reagent, aspirated into the tip, in eitherorder. Any such solution can be used. A highly preferred examplecomprises a 3% bovine serum albumin in a 0.1 molar phosphate bufferedsaline solution containing anti-B IgM clones formulated from tissueculture supernatant (1, 20, and 31 μg/ml concentrations) plus 0.004%FD&C blue dye number 1. All concentrations are % by weights.

[0091] It is not necessary that the detection of a strong, weak, ornegative reaction of such blood typing be done outside of the mixingtip. Instead, it can be achieved by detecting the amount ofagglutination separation within the tip, and thus the strength of theblood typing reaction.

[0092] Turning to FIG. 8, this detection is preferably done by scanningfor absorbance or light scattering at a position in narrower tip portion114″. (Any other embodiment of the invention can also be used.) That is,at the position of arrow 300, an appropriate optics such as aconventional fiber optics is used to deliver light of a predeterminedwavelength that is then transmitted into the tip. The amount of lightthat is absorbed, measured approximately 10 minutes after mil has beencompleted, is then detected as shown schematically by arrow 302, or theamount of light scattered is detected as shown by arrow 304. The resultsdiffer depending on how much agglutination has occurred, as shown below.If absorbance is used, suitable wavelengths include 540 nm, and/or 830nm. The former is particularly useful since that is the peak absorptionof hemoglobin. Detection of the amount of light scattered, as at 304, isparticularly useful to avoid interference from any hemolysis.

[0093]FIGS. 10 and 11 illustrate the method using absorbance and anilluminating wavelength of 540 nm. In the case of FIG. 10, the liquid ispassed down from portion 118′″ to portion 114′″, after 10 minutes havepassed after it has been mixed sufficiently. At the zero to about 18% ofthe volume that passes, the amount of absorbance rises from zero due tothe passage of air. After that, only liquid is passed by the scanner,and the first part of that liquid is very absorbent, regardless ofwhether the reaction is negative, weak, or strong. However, after about50% of the liquid has been scanned, the results deviate depending on theamount of agglutination achieved. A strong reaction clumps the red cellsso well that after about 65%, the volume is essentially free of cellsand is clear. A weak reaction has less absorption, but still much morethan the strong, after 65% of the volume scanned.

[0094] Alternatively, the liquid can be moved upward from portion 165′″into narrower portion 114′″ and on upward into portion 118′″, to do thescanning. The results are shown in FIG. 11. Differentiation of theresults occurs when from zero to 18% of the volume has been scanned.That is, the first portion to flow past the scanner is the liquidportion free of red cells, in the event of a strong reaction, becausealmost all of the cells have coagulated together. But in the case of theweak reaction, some red cells are still unagglutinated and remain inthat first portion of the volume, as shown by the middle curve of FIG.11.

[0095] It is not only blood typing agglutination that is useful as anagglutination reaction in the tip of the invention. Agglutination causedby a coagulating reagent allows separation of the cellular fraction ofwhole blood from the plasma, to occur in the tip. That is, when theagglutinating reagent is selected from conventional coagulating reagentssuch as a polyelectrolyte, eg, polylysine, or an antibody such asanti-glycophorin, the mixing within the tip as described above will notonly cause coagulation of all the red cells, but it will also lead to aphysical separation of those coagulated cells from the plasma. The cellssettle to the bottom of the tip, e.g., tip portion or cavity 165′″ ofFIG. 9. At this juncture, those cells can then be expelled by dispensingthem out of the orifice of the tip, leaving only plasma remainingbehind. That plasma can then be dispensed onto a suitable platform fortesting, for example, into a well or cup adapted for immunoassay, suchas is described in U.S. Pat. No. 5,441,895.

[0096] The following are non-limiting working examples of the mixingsteps of this invention:

Example No. 1

[0097] A probe was constructed having two capillaries with differentinner diameters. The smaller capillary had an inner diameter of 0.557mm. The larger capillary had an inner diameter of 2.29 mm. The length ofthe smaller capillary was 41 mm, which holds up to 10 micro-liter offluid The larger capillary had a length of 30

[0098] A type B blood of 4 micro-liters was aspirated from the bottomend of the small capillary by the pump. The pump then continued towithdraw 1 micro-liters of air in the small capillary. 4 micro-liters ofthe agglutinating reagent described above was aspirated thereafter andthe air bubble separated the two liquids in the smaller capillary.

[0099] The pump was then driven to move all the fluids across thetransition zone between the small and large capillary with a flow rateof 0.5 micro-liter/second. Once in the larger capillary, a spherical airbubble was created by the surface tension, and the two liquids startedto encounter and mix. As the pump drove the fluids to flow down into thesmaller capillary with a flow rate of 0.5 micro-litter/second, thebubble was eliminated.

[0100] The mixture of the two fluids was oscillated between the twocapillaries with a constant flow rate of 0.5 micro-liters/second. Theagglutinated structure formation was visible at the end of the fistcycle of this motion. Phase separation was very significant at the endof the second cycle in the small capillary, with clear supernatant inthe up portion and the agglutinated cell structure in the bottomportion. Some very small agglutinated cells were still visible in thesupernatant at this stage. The phase separation was completed by the endof the third cycle, with almost zero cell structure left in thesupernatant.

[0101] The total time period for the three cycles was 2 minutes. Weakerreactions can be expected to take longer.

[0102] Once complete mixing has been achieved, it is then necessary, ofcourse, to achieve a determination of the blood type from theagglutinated results. Although that is not part of this invention, onemethod of doing this is to make a visual observation of lighttransmittance through the mixture to determine the amount ofagglutination within a fixed time of the agglutination reaction. A chartis used for comparison, and the user estimates the blood type from theamount of clumping or agglutination observed in whichever probe portionthat the combined liquids are in at the time.

Example2

[0103] A probe was constructed similar to shown in FIG. 9, except thatthe tip portion 165′″ was cut off at line C-C to create a cone-shapedtip portion having an inside diameter at D₃ of about 2.54 mm, and at cutline C-C of about 1 mm, with a cone angle of about 20 degrees and alength of 10 mm. Tip portion 114′″ had a length of about 15 mm and aninside diameter D₁ of about 1 mm. Tip portion 118 ′″ had a diameter D₂of about 4.7 mm. Blood in an amount of 10 microliters was aspirated intothe entire tip ensemble through cone portion 165′″, after which the conewas wiped clean. Then 10 microliters of reagent were aspirated into thetip in the same manner, producing a total liquid volume of 20microliters. This total volume was then moved back and forth so as toproceed entirely into portion 118′″ and then entirely into portion165′″, and so forth, until mixing was complete. This required 7.5repetitions (cycles) at a flow rate of 50 microliters per sec. Totaldisplacement of fluids was 40 microliters in each direction of motion,and the time required for complete mixing was about 15 sec.

[0104] The invention disclosed herein may be practiced in the absence ofany element which is not specifically disclosed herein.

[0105] The invention has been described in detail with particularreference to preferred embodiments thereof but it will be understoodthat variations and modifications can be effected within the spirit andscope of the invention.

What is claimed is:
 1. In a method of mixing a plurality of liquidscomprising the steps of: a) providing a probe tip with an internalcavity having a plurality of different inside diameters; b) providing byaspiration a plurality of liquids inside a portion of the probe tip; c)moving at least most of said liquids back and forth at least severaltimes between a part of said cavity with a smaller inside diameter and apart with a larger inside diameter, said larger and smaller diametersbeing sufficient to provide a sufficient rotation of liquid as it movesbetween diameters to cause mixing of said liquids; the improvementwherein the capillary number resulting from the mixing in said step c)does not exceed about 0.01, said capillary number being defined as theratio of liquid velocity times viscosity and surface tension, so thatany tails formed during said mixing step c) are minimized.
 2. A methodas defined in claim 1, wherein the capillary number of step c) does notexceed about 0.001, so that any entrained air bubble is more readilyremoved from said liquids as they are mixed.
 3. A method as defined inclaim 2, wherein said any bubble is aspirated into said probe tipin-between said plural liquids, with a volume that is less than thatwhich prevents mixing of the liquids in the part of said cavity havingthe larger of said inside diameters.
 4. In a method of mixing aplurality of liquids comprising the steps of: a) providing a probe tipwith an internal cavity having a plurality of different insidediameters; b) providing by aspiration a plurality of liquids inside aportion of the probe tip; c) moving at least most of said liquids backand forth at least several times between a part of said cavity with asmaller inside diameter and a part with a larger inside diameter, saidlarger and smaller diameters being sufficient to provide a sufficientrotation of liquid as it moves between diameters to cause mixing of saidliquids; the improvement wherein said cavity parts comprise two separatebut matable tip portions, and said method further includes the step ofmounting a mountable tip portion of one of said inside diameters ontosaid tip portion of the other inside diameter in-between aspiration ofliquids, such that carry-over contamination between liquids isprevented.
 5. A method as defined in claim 4, and further including thesteps of removing said tip portion after each additional liquid isaspirated, and attaching a new tip portion before aspirating into saidprobe tip an additional liquid.
 6. A method as defined in claim 4,wherein said mountable tip portion has a larger inside diameter tan thatof said tip portion on which it is mounted.
 7. A method as defined inclaim 6, wherein said tip portion on which said mountable portion ismounted, further includes two inside diameters of significantlydifferent values, so that flow of said liquids past a demarcation zonebetween said differently valued inside diameters also providesrotational mixing of the liquids.
 8. A method as defined in claim 7,wherein the larger of said differently valued inside diameters is atleast as large as the largest inside diameter of said mountable tipportion.
 9. A method as defined in claim 8, wherein said larger of saiddifferently valued diameters is at least equal to three times the valueof the smaller of said differently valued inside diameters.
 10. A methodas defined in claim 6, wherein the largest of said inside diameter ofsaid mountable tip portion is at least equal to three times the value ofthe smaller of said differently valued inside diameters.
 11. In a methodof mixing a plurality of liquids comprising the steps of: a) providing aprobe tip with an internal cavity having a plurality of different insidediameters; b) providing by aspiration a plurality of liquids inside aportion of the probe tip; c) moving at least most of said liquids backand forth at least several times between a part of said cavity with asmaller inside diameter and a part with a larger inside diameter, saidlarger and smaller diameters being sufficient to provide a sufficientrotation of liquid as it moves between diameters to cause mixing of saidliquids; the improvement wherein said inside diameters are each ameasure of a cross-sectional flow-through area of said cavity part, andthe cross-sectional flow-through area of said larger inside diameter isat least three times the cross-sectional flow through area of saidsmaller inside diameter.
 12. A method as defined in claim 11, whereinsaid one liquid is whole blood and wherein said moving step causes onlymixing such that cells that have agglutinated are less likely to breakapart.
 13. In a method of mixing a plurality of liquids comprising thesteps of: a) providing a probe tip with an internal cavity having aplurality of different inside diameters; b) providing by aspiration aplurality of liquids inside a portion of the probe tip; c) moving atleast most of said liquids back and forth at least several times betweena part of said cavity with a smaller inside diameter and a part with alarger inside diameter, said larger and smaller diameters beingsufficient to provide a sufficient rotation of liquid as it movesbetween diameters to cause mid of said liquids; the improvement whereinsaid larger inside diameter is obtained by i) selecting as a first tipportion a tapered tip at least a portion of which has an inside diameterthat is much larger than the smaller inside diameter of the probe tip,and ii) joining said tapered tip to said probe tip having the smallerinside diameter using a joining collar mounted around said tip portionof step b).
 14. In a method of mixing a plurality of liquids comprisingthe steps of: a) providing a probe tip with an internal cavity having aplurality of different inside diameters; b) providing by aspiration aplurality of liquids inside a portion of the probe tip; c) moving atleast most of said liquids back and forth at least several times betweena part of said cavity with a smaller inside diameter and a part with alarger inside diameter, said larger and smaller diameters beingsufficient to provide a sufficient rotation of liquid as it movesbetween diameters to cause m of said liquids; the improvement whereinthe total amount of liquid provided by said step b) is such that if allliquid is moved into said part with the larger inside diameter, thelarger inside diameter is greater than the height of the total movedliquid, but less than twice the height of the total moved liquid, sothat mixing as per step c) is maximized.
 15. A method as defined inclaim 14, wherein said mixing is accomplished without any substantialagitation or shaking of the probe tip.
 16. In a method of mixing aplurality of liquids comprising the steps of: a) providing a probe tipwith an internal cavity having a plurality of different insidediameters; b) providing by aspiration a plurality of liquids inside aportion of the probe tip, c) moving at least most of said liquids backand forth at least several times between a part of said cavity with asmaller inside diameter and a part with a larger inside diameter, saidlarger and smaller diameters being sufficient to provide a sufficientrotation of liquid as it moves between diameters to cause mixing of saidliquids; the improvement wherein said step c) comprises moving at leastmost of the liquids back and forth at least between said cavity partwith said smaller inside diameter and a part of said cavity of a largerinside diameter located at opposite ends of said cavity part of saidsmaller inside diameter, so that mixing efficiency is enhanced byrotation of the liquid as it moves past said opposite ends, rather thana single end of said smaller inside diameter cavity part.
 17. A methodas defined in claim 16, wherein said liquids are completely mixed within7.5 repetitions of said movement back and forth at a flow rate of about50 microliters per sec., within about 10 sec.
 18. A probe tip for mixingliquids within the tip after aspiration of the liquids therein to, saidtip comprising a wall defining 3 connected cavities of unequal insidediameters one of the compartments being sandwiched as a middlecompartment between the other two which form end compartments, each twoadjacent cavities being connected by a transition zone wall and saidinside diameters being sufficiently unequal in said adjacent 2 cavitiesas to cause rotational mixing of liquids as they move past saidtransition zone wall, wherein said transition zone of the one cavity isformed by a variance of said inside diameter that increases in value asthe middlemost cavity is transited outward into either of said other twoend cavities.
 19. A probe as defined in claim 18, wherein one of saidend cavities is defined by a wall portion removably mounted on a walldefining said middle cavity.
 20. A probe as defined in claim 19, whereinthe inside diameter of at least one of said end cavities is at leastequal to three times the value of the smaller of said differently valuedinside diameters.
 21. A method of determining the strength of anagglutination reaction within a hollow container comprising wallscapable of transmitting light at certain predetermined wavelengths,comprising the steps of: a) providing a mixture of a sample and anagglutinating reagent within a first cavity of the container, saidcavity having a first inside diameter, b) transferring the mixture to asecond cavity having a second inside diameter substantially smaller thansaid first inside diameter, c) scanning the liquid within said secondcavity during said step b) with a beam of light at said predeterminedwavelengths, said 10% portion being that portion closest to said firstcavity, d) after said scan step c), detecting the amount of lightabsorbed within or scattered by said 10% portion by said beam, e)transferring said mixture back into said first cavity, f) repeatingsteps b)-d) at least once until some agglutinated material has separatedfrom non-agglutinated material, and g) calculating the amount ofagglutination from the absorbance or scattering detected in said stepd).
 22. A method as defined in claim 21, wherein said transfer stepmoves the liquid down from the first cavity to said second cavity, sothat gravity assists in said separation of step f).
 23. A method asdefined in claim 22, wherein said step g) comprises determining whatpercentage of the total possible absorbance is detected at a preselectedpercent of the volume scanned that is indicative of agglutinatingreactions, as an indication of the % and therefore the strength, of theagglutination that has occurred.
 24. A method as defined in claim 21,wherein said detecting step d) uses radiation at about 540 nm, the peakabsorption wavelength of hemoglobin.
 25. A method as defined in claim21, wherein said step d) comprises detecting the amount of scatteredradiation, so that any hemolysis interference is avoided.
 26. A methodof agglutinate blood cells in whole blood, comprising the steps of a)aspirating whole blood into a disposable tip mounted on a probe, saidtip having at least two portions with significantly different insidediameters, connected to each other by a transition zone, b) aspiratinginto the same tip thereafter, an agglutinating reagent, and c) movingsaid blood and reagent back and forth as a total liquid, first entirelyinto one of said portions and then entirely into the other of saidportions, a sufficient number of times so as to cause coagulation of thecells of the whole blood, and then subsequent separation of plasma fromthe coagulated cells.
 27. A method of separating as defined in claim 26,wherein said cells are allowed to settle adjacent to an exit orifice ofsaid tip, and d) thereafter, dispensing said cells out of said tip,leaving only plasma remaining therein.
 28. A method of separating asdefined in claim 27, and further comprising the step of e) dispensing atleast a portion of said remaining plasma from said tip into a reactionwell adapted for carrying out an immunoassay of the plasma.
 29. A methodof separating as defined in claim 26, wherein said agglutinating reagentis a polyelectrolyte or an antibody.